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Characterization of Burkholderia glumae Putative Virulence Factor 11 (PVF11) via Yeast Two-Hybrid Interaction and Phenotypic Analysis
Plant Pathol. J. 2019;35:280-286
Published online June 1, 2019
© 2019 The Korean Society of Plant Pathology.

Juyun Kim1,2,†, Namgyu Kim1,2,†, Mohamed Mannaa1,2, Hyun-Hee Lee1,2, Jong-Seong Jeon3, and Young-Su Seo1,2,*

1Department of Microbiology, Pusan National University, Busan 46241, Korea, 2Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea, 3Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea
Correspondence to: *Corresponding author: Phone) +82-51-510-2267, FAX) +82-51-514-1778
E-mail) yseo2011@pusan.ac.kr
Received February 7, 2019; Revised March 20, 2019; Accepted March 20, 2019.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

In this study, PVF11 was selected among 20 candidate pathogenesis-related genes in Burkholderia glumae based on its effect on virulence to rice. PVF11 was found to interact with several plant defense-related WRKY proteins as evidenced through yeast-two hybrid analysis (Y2H). Moreover, PVF11 showed interactions with abiotic and biotic stress response-related rice proteins, as shown by genome-wide Y2H screening employing PVF11 and a cDNA library from B. glumae-infected rice. To confirm the effect of PVF11 on B. glumae virulence, in planta assays were conducted at different stages of rice growth. As a result, a PVF11-defective mutant showed reduced virulence in rice seedlings and stems but not in rice panicles, indicating that PVF11 involvement in B. glumae virulence in rice is stage-dependent.

Keywords : Burkholderia glumae, virulence, yeast two hybrid analysis
Supplemental Materials
Figures
Fig. 1. Yeast two hybrid (Y2H) assays for Burkholderia glumae PVF11 interaction with rice defense-related proteins. (A) Positive Y2H interaction between PVF11, as prey, and WRKY transcriptional factors, as baits. The empty bait vector was used as a negative control. (B) Positive Y2H interaction between PVF11, as bait, and rice cDNA libraries (Y28, Y29 and Y34) as prey. In both Y2H assays, positive interactions are indicated by hybridized yeast cell survival on LTH (SD/-His/-Leu/-Trp) and LTHA (SD/-Ade/-His/-Leu/-Trp) agar plates.
Fig. 2. Pathogenicity assays of Burkholderia glumae BGR1 wild-type, PVF11 defective mutant, and PVF11C complemented mutant in rice plants at different growth stages (seedling, vegetative, and reproductive). (A) Photographs from the seedling assay showing a clear deleterious effect of wild-type BGR1 treatment and reduced virulence in the ΔPVF11 mutant-treated seedlings. The complemented mutant, PVF11C, apparently recovered the wild-type virulence. (B) Bar graphs represent the shoot and root lengths as a measure of the bacterial virulence to the seedlings. The different uppercase and lowercase letters on the error bars represent significant differences between shoot and root lengths, respectively, with P < 0.05, according to least significant difference test (LSD). (C) Photographs of rice stems at the vegetative stage showing the typical blight symptoms of infected rice stems. Treatment with the ΔPVF11 mutant resulted in reduced virulence as limited symptomatic lesions were observed, compared to plants infected with wild-type BGR1 or the complemented mutant PVF11C. Red arrows show the spread of symptomatic lesions away from the inoculation sites. (D) Bar graphs showing the bacterial populations in treated stems 8 days after inoculation, expressed as log colony forming units (cfu)/g rice stem. The different letters on the error bars indicate significant differences according to LSD test (P < 0.05). (E) Photographs from the reproductive stage assay on rice panicles showing the typical blight symptoms on the rice grains after treatment with BGR1, ΔPVF11, and PVF11C. (F) Stacked bar graphs representing disease severity distribution pattern on the harvested grains. There was no difference in disease severity between the wild-type, ΔPVF11, and PVF11C mutants. Sterile distilled water was used as negative control in all assays.
Tables

Putative encoded products and ontology of genes from infected rice that showed positive interaction with PVF11

Clone No. Locus ID Gene Product Gene ontology
Y28 LOC_Os04g57200 Heavy metal transport/detoxification protein, putative, expressed Metal ion binding and transport
Detoxification
Y29 LOC_Os08g09200 Aconitate hydratase protein, putative, expressed Aconitate hydratase
Catalytic activity
DNA binding
Endogenous stimulus response
Biotic and abiotic stress response
Y34 LOC_Os01g12910 Thioesterase family protein, putative, expressed Hydrolase activity
Inhibition to ROS
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